Introducing heterologous genes into animal host cells and screening for expression of the added genes is a lengthy and complicated process. Typically a number of hurdles have to be overcome: (i) the construction of large expression vectors; (ii) the transfection and selection of clones with stable long-term expression, eventually in the absence of selective pressure; and (iii) screening for high expression rates of the heterologous protein of interest.
1. Selection of Clones Expressing the Heterologous Gene
Selection of the clones having integrated the gene of interest is performed using a selection marker conferring resistance to a selective pressure. Most of the selection markers confer resistance to an antibiotic such as, e.g., neomycin, kanamycin, hygromycin, gentamycin, chloramphenicol, puromycin, zeocin or bleomycin.
When generating cell clones expressing a gene of interest from expression vectors, host cells are typically transfected with a plasmid DNA vector encoding both the protein of interest and the selection marker on the same vector. Quite often the capacity of a plasmid is limited and the selection marker has to be expressed from a second plasmid, which is co-transfected with the plasmid comprising the gene of interest.
Stable transfection leads to random integration of the expression vector in the genome of the host cell. Use of selective pressure, e.g. by administrating an antibiotic to the media, will eliminate all cells that did not integrate the vector containing the selection marker providing resistance to the respective antibiotic or selective pressure. If this selection marker is on the same vector as the gene of interest or, if this selection marker is on a second vector and vector comprising the gene of interest was co-integrated, the cells will express both the selection marker and the gene of interest. It is frequently observed, however, that the expression level of the gene of interest is highly variable depending on the site of integration.
Furthermore, when removing selective pressure, expression becomes quite often very unstable or even extinguished. Only a small number of initial transfectants are thus providing high and stable long-term expression and it is time-consuming to identify these clones in a large population of candidates. Typically, high expressing candidates are isolated and then cultivated in absence of selective pressure. Under these conditions a large proportion of initially selected candidates are eliminated due to their loss of gene of interest expression upon removal of selective pressure. It would thus be advantageous to cultivate the candidates, following an initial period of selection for stable transfection, in absence of selective pressure and only then screen for gene of interest expression.
2. Screening for High Expressing Clones
Screening for high-expressing clones for a protein of interest is often done by methods directly revealing the presence of high amounts of the protein. Typically immunologic methods, such as ELISA or immunohistochemical staining, are applied to detect the product either intracellularly or in cell culture supernatants. These methods are tedious, expensive, time-consuming, and often not amenable to high throughput screenings (HTS). In addition, an antibody reactive to the expressed protein must be available.
Attempts to quantify the protein amounts by Fluorescence-Activated Cell Sorting (FACS) have also been made, but only with a limited success, especially in the case of secreted proteins (Borth et al., 2000)
One approach for the screening of high expression rates of the protein of interest would be the use of an easily measurable surrogate marker, expressed from the same vector as the gene of interest (Chesnut et al., 1996). The idea underlying the use of a measurable surrogate marker is that there is a correlation between the expression of the gene of interest and the surrogate marker gene due to the physical link of the two genes on the same vector.
Numerous easily measurable markers are available in the art. They usually correspond to enzymes, which act on a chromogenic or luminogenic substrate such as, e.g., the β-glucuronidase, the chloramphenicol acetyltransferase, the nopaline synthase, the β-galactosidase, secreted alkaline phosphatase (SEAP) and the DHFR. The green fluorescent protein (GFP) may also be used as a measurable marker in FACS. The activity of all these proteins can be measured by standard assays that may be used in HTS.
The drawback of this approach is the use of yet another expression cassette for the surrogate marker gene. This renders the expression vector rather bulky, hosting expression units comprising a promoter, a cDNA and polyadenylation signals for at least three proteins (i.e., the gene of interest, the selection marker and the surrogate marker). For multi-chain proteins the situation becomes even more complex. Alternatively, individual plasmid vectors expressing the three genes, which encode the protein of interest, the selection marker and the surrogate marker respectively, could be co-transfected. However, it is likely that the vectors would be either integrated at different loci, or exhibit varying and uncorrelated expression.
A promising approach for overcoming the above limitations consists in the use of a chimeric marker that combines the functional properties of a selection marker and of a measurable marker.
Such bifunctional markers have been described by, e.g., Bennett et al. (1998), Imhof and Chatellard (2006) and Dupraz and Kobr (2007). Bennett et al. (1998) disclose the GFP-ZeoR marker, which confers resistance to Zeocin antibiotic, which corresponds to a fusion protein between the Green Fluorescent Protein (GFP) and a protein conferring resistance to zeocin. Imhof and Chatellard (2006) disclose the Lupac marker, which corresponds to a fusion between the firefly luciferase protein and a protein conferring resistance to puromycin. Dupraz and Kobr (2007) discloses the PuroLT marker, which corresponds to a fusion protein between the synthetic peptide described by Griffin et al. (1998) and a protein conferring resistance to puromycin.
Miller et al. (2005), in an article showing that fluorescent TMP is an alternative to fluorescent MTX, discloses a fusion protein between a protein conferring resistance to puromycin and a DHFR of bacterial origin. DHFR is used as measurable marker that can be detected by binding to fluorescent MTX or to fluorescent TMP. This article envisions the use of the fusion protein in the field of siRNA gene silencing.
Hence, all markers available for the selection of clones expressing high levels of a recombinant protein correspond to bifunctional markers, which confer resistance to a single toxic compound.
In addition to the bifunctional marker, the vectors used for generating high producer clones usually comprise an amplifiable gene that leads to an increase in copy number when under selective pressure. The copy number of a gene of interest positioned adjacent to the amplifiable gene will also increase, thus leading to the establishment of clones expressing high levels of the protein of interest (Kaufman et al., 1985; Kaufman et al., 1986; Kim et al., 2001; Omasa, 2002). Commonly used amplifiable genes include e.g. dihydrofolate reductase (DHFR), Glutamine synthetase (GS), multiple drug resistance gene (MDR), ornithine decarboxylase (ODC), adenosine deaminase (ADA) and N-(phosphonoacetyl)-L-aspartate resistance (CAD).
The finding of a novel and powerful chimeric surrogate marker, conferring resistance to more than one toxic compound and also allowing gene amplification, would be extremely useful in the field of industrial production of therapeutic proteins.